We have made significant progress toward our goals: 1. DISRUPTION OF NUCLEOSOMES DURING HEAVY TRANSCRIPTION. Cole HA, Ocampo J, Iben JR, Chereji RV, Clark DJ Paired-end sequencing performed by Dr Jun Zhu's Lab (NHLBI). Eukaryotic chromatin is composed of nucleosomes, which contain nearly two coils of DNA wrapped around a central histone octamer. The octamer contains an H3-H4 tetramer and two H2A-H2B dimers. Gene activation is associated with chromatin disruption: a wider nucleosome-depleted region (NDR) at the promoter and reduced nucleosome occupancy over the coding region. We have examined the nature of disrupted chromatin after induction, using MNase-seq to map nucleosomes and sub-nucleosomes, and a refined high-resolution ChIP-seq method to map histones H4 and H2B, and RNA polymerase II (Pol II) genome-wide. Over coding regions, induced genes show a differential loss of H2B relative to H4, which correlates with Pol II density and the appearance of sub-nucleosomes. After induction, Pol II is surprisingly low at the promoter, but accumulates on the gene and downstream of the termination site, implying that dissociation is slow. Thus, induction-dependent chromatin disruption reflects both eviction of H2A-H2B dimers from nucleosomes and the presence of queued Pol II elongation complexes. We propose that slow Pol II dissociation after transcription is a major factor in chromatin disruption and that it may be of critical importance in gene regulation. Currently, we are testing this hypothesis. Cole HA, Ocampo J, Iben JR, Chereji RV, Clark DJ (2014). Heavy transcription of yeast genes correlates with differential loss of histone H2B relative to H4 and queued RNA polymerases. Nucleic Acids Res., 42, 12512-12522 (PMID 25348398). 2. INTERPLAY AMONG DIFFERENT ATP-DEPENDENT CHROMATIN REMODELING MACHINES DETERMINES NUCLEOSOME SPACING ON ACTIVE AND INACTIVE GENES. Ocampo J, Chereji RV, Eriksson PR, Clark DJ. Paired-end sequencing performed by Dr Jun Zhu's Lab (NHLBI). In yeast, most genes have a nucleosome-depleted region (NDR) at the promoter and an array of regularly spaced nucleosomes that is phased relative to the transcription start site. We addressed the roles of four remodeling machines (ISW1, ISW2, CHD1 and RSC) in specifying this chromatin organization. We sequenced nucleosomes from isogenic strains with the essential RSC8 gene linked to a GAL promoter and isw1, isw2 or chd1 null mutations in all combinations. Globally, RSC depletion results in a shift of the array into the NDR, isw1 shows reduced spacing and both isw1 and chd1 show weakened phasing, whereas isw2 has no effect. Generally, chromatin organization in the multiple mutants is the sum of the effects observed in the single mutants, indicating that these remodelers are not redundant but have distinct functions. Genes in wild type cells display a wide range in nucleosome spacing; transcriptionally active genes tend to exhibit extreme spacing. Our data suggest that, on active genes, RSC determines the location of the +1 nucleosome, which is used as a reference by the spacing enzymes, ISW1 and CHD1, to restore the nucleosomal array after transcription. Phasing on inactive genes also requires ISW2, which antagonizes RSC by shifting nucleosomal arrays in the opposite direction. Furthermore, inactive genes have longer spacing and are enriched in linker histone, suggesting that they may have more condensed chromatin than active genes. Manuscript submitted. 3. NOVEL NUCLEOSOMAL PARTICLES CONTAINING CORE HISTONES AND LINKER DNA BUT NO HISTONE H1. Cole HA, Cui F, Ocampo J, Burke TL, Nikitina T, Nagarajavel V, Kotomura N, Zhurkin VB, Clark DJ. Paired-end sequencing performed by Dr Jun Zhu's Lab (NHLBI). Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining the accessibility of control regions. The nucleosome contains 147 bp of DNA wrapped 1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and 160 bp, and then converts it to a core particle, containing 147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are observed in yeast lacking H1 and in H1-depleted chromatin from mouse liver. They can be reconstituted in vitro using just purified recombinant yeast core histones or native chicken core histones and DNA. We propose that these proto-chromatosomes are fundamental subunits of chromatin, which specify the topography of the H1 binding site and may influence nucleosome spacing independently of H1. Manuscript in revision. 4. GENOME-WIDE COOPERATION BY HAT Gcn5, REMODELER SWI/SNF, AND CHAPERONE Ydj1 IN PROMOTER NUCLEOSOME EVICTION AND TRANSCRIPTIONAL ACTIVATION. Qiu H, Hu C, Chereji R, Cole HA, Rawal Y, Clark DJ, Hinnebusch AG. Paired-end sequencing performed by Dr Jun Zhu's Lab (NHLBI). Gene activation generally involves nucleosome removal and/or nucleosome shifts in the promoter region. The process of nucleosome assembly and disassembly at promoters involves histone chaperones, chromatin remodeling complexes and histone acetyltransferases. We asked whether these co-factors function ubiquitously and what effect nucleosome eviction has on transcription genome-wide. We performed chromatin immunoprecipitation (ChIP-seq) experiments to detect histone H3 and Pol II in mutants lacking single or multiple co-factors (the SWI/SNF remodeling complex, the Gcn5 histone acetyltransferase and the Ydj1 histone chaperone). We focused on 200 genes regulated by the transcription activator Gcn4. After induction, 70 of these genes show significantly reduced H3 occupancy at their promoters. All three of the co-factors examined play a role in H3 eviction at most, but not all Gcn4 target promoters, with Gcn5 playing the greatest role and Ydj1 the least. In fact, the three co-factors cooperate to various extents in H3 eviction at virtually all yeast promoters. Reduced H3 eviction in co-factor mutants is correlated with reduced Pol II occupancies on Gcn4-regulated genes and on the most highly expressed constitutive genes, but the relative Pol II levels at most genes are unaffected. Our observations indicate that promoter nucleosome eviction is important for heavy transcription of highly expressed genes, but that other steps in gene activation are more important for most other yeast genes. Manuscript submitted.